The fluorescence or luminescence lifetime is the average time that a fluorochrome spends in the excited state. The sensitivity of the fluorescence (or luminescence) lifetime to environmental factors can be exploited to investigate the physico-chemical environment of fluorochromes. From the early 1990s, fluorescence lifetime imaging microscopy (FLIM) allowed the quantitative and robust imaging of pH, ion concentration, oxygen content, etc., in living cells, tissues and model organisms. Moreover, FLIM enabled protein-protein interactions to be mapped in cells, by imaging the occurrence of the phenomenon of Förster Resonance Energy Transfer (FRET).
However, even given its intrinsic value, the spreading of FLIM instrumentation in laboratories is limited by its cost and by the required know-how necessary for its maintenance and operation. More recently, the use and value of FLIM for diagnostic applications, histology and screening has been demonstrated. Thus, the establishment of cost-effective and user-friendly systems is a requirement to allow its wide-spread application in laboratories and research fields.
Both the detection of lifetimes in the time-domain (TD) and in the frequency-domain (FD) regime, and the use of laser scanning (LSM) and wide-field microscopy have been widely described in literature. In the time domain, the donor molecule is excited with a pulsed light source and the fluorescence intensity decay is recorded as a function of time. Because the fluorescence lifetimes of typically used organic and genetic fluorophores range in the low nanosecond region, pico- or femto-second pulsed sources and repetition rates in the megahertz range are required. These light sources can also be used in the frequency domain. More often, FD uses sinusoidally modulated light sources. FD detection allows the use of higher illumination duty cycles, thereby permitting the fastest acquisition times. The major advances in solid-state technologies today enable the use of cost-effective laser diodes (LDs) and inexpensive light emitting diodes (LEDs) as directly modulated light sources. The former can be pulsed in the pico-second range, albeit at the cost of reduced laser operation lifetime and subsequent operation costs. LEDs can be pulsed in the nanosecond range, sufficient to obtain fluorescence lifetime information, but are not ideal for the robust and efficient measurement of nanosecond or sub-nanosecond decays. On the other hand, both LDs and LEDs can be intensity-modulated in the MHz region, the optimal frequency range for the frequency-domain detection of nanosecond decaying fluorochromes. Luminescent probes exhibit lifetimes in the microsecond to millisecond range, thus posing less stringent technological requirements. LDs and LEDs are increasingly replacing expensive continuous-wave lasers that are modulated by external optical devices, and mode-locked lasers.
The detectors used for laser-scanning and wide-field microscopes differ substantially (Esposito A. and Wouters F. S., “Fluorescence Lifetime Imaging Microscopy”, In: Current Protocols in Cell Biology; Bonifacino J. S., Dasso M., Harford J. B., Lippincott-Schwartz J., and Yamada K. M., editors, 2004). The former use sensitive and fast point detectors like photo-multiplier tubes (PMTs), multi-channel-plate PMTs (MCP-PMTs), avalanche photodiodes (APD) or single photon counting APDs (SPADs) in the various detection techniques: time-correlated single-photon-counting (TCSPC) and time-gated (TG) detection in the TD, and cross-correlation for FD operation. Wide-field detection requires the use of a spatially-resolved MCP detector. MCPs are relatively expensive, prone to photo-damage and require elaborate electronics for operation. Moreover, although the time properties of MCPs are optimal, their spatial resolution is relatively low, they can present “chicken-wire” artifacts caused by the fiber-optic coupling to CCD pixels, and can inject a relatively high noise level in the measurement. For these reasons, a robust solid-state detector presents a desirable alternative to multi-channel-plates for routine FLIM application by a larger user community and also for application in medical diagnostics and high-throughput pharmacological compound screening. Therefore, the detector of the invention does not require the use of a multi-channel-plate for the signal demodulation.
The sensitivity or photoeconomy of FLIM systems has been described by the use of the ratio (F value) of the coefficient of variation of the lifetime image and the intensity map. The combination of a scanning system and femtosecond-laser sources with detection in the time domain (TCSPC) or frequency domain (lock-in) has been shown to have F values close to unity, i.e., the maximally achievable photoeconomy. The use of an MCP for wide-field detection deteriorates this performance. Under sine-ve illumination and detection with three images at different phases, the F values reach a value higher than 7, i.e., approx. 50 times more photons have to be collected to achieve a signal-to-noise ratio equal to a system that uses a femtosecond laser and an MCP-PMT as the detector system. A lock-in imager can offer higher sensitivity than an MCP. The combination of solid-state technologies, frequency-domain detection and wide-field microscopy seems to be a good compromise in terms of cost and simplicity of use.
In the recent past, pioneering works have been published (Mitchell A. C., Wall J. E., Murray J. G., and Morgan C. G., “Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging”, Journal of Microscopy, Vol. 206, Pt. 3, 225-232, 2002; Mitchell A. C., Wall J. E., Murray J. G., and Morgan C. G. “Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera”, Journal of Microscopy, Vol. 206, Pt. 3, 233-238, 2002; Nishikata K., Kimura Y., Takai Y., Ikuta T., and Shimizu R., “Real-time lock-in imaging by a newly developed high-speed image-processing charged coupled device video camera”, Review of Scientific Instruments, 1393-1396, 2003) that aimed at the use of charged-coupled-devices (CCDs) in FLIM. They reported on the possible modification of a commercial CCD camera to allow lifetime detection. Their theoretical upper limit was estimated at approximately 10 MHz, but a realistic practical implementation was demonstrated to be feasible at a lower modulation frequency of 500 kHz. These modulation frequencies are sub-optimal for use with typically used fluorophores in life sciences.
By the same token, EP-1′162′827 A2 teaches the use of CCDs for time-resolved measurements of luminescence. For this purpose, conventional CCD arrays are described in which charges are moved bi-directionally from one pixel to another, or in which the spatial extent of charge collection from pixels is controlled.
On the other hand, CCD lock-in imagers are commercially produced for time-of-flight (TOF) ranging (Oggier T., Lehmann M., Kaufmann R., Schweizer M., Richter M., Metzler P., Lang G., Lustenberger F., and Blanc N., “An all-solid-state optical range camera for 3D real-time imaging with sub-centimeter depth resolution (SwissRanger™)”, Proc. of SPIE Vol. 5249, 534-545, 2004). The field of TOF ranging is completely different from the field of microscopy. The former deals with large objects lying far away (in distances of several meters or more); knowledge of electric engineering and microelectronics is required. In the latter, small objects lying under a microscope are investigated, for which experiences in life sciences and in optics are needed. To date, these two technical fields have not overlapped.